Dual RNA 3’ end processing of H2A.X messenger RNA maintains DNA damage repair throughout the cell cycle

Replication-dependent (RD) histone mRNA are the only non-polyadenylated protein coding transcripts in humans. However, a subset of RD histone genes also expresses polyadenylated mRNA upon terminal differentiation to maintain histone proteins. We now show that even in cycling cells, RD histone genes express some poly(A) mRNA. The H2AFX gene, encoding the DNA damage signalling histone variant H2A.X, expresses both an S-phase-specific SL mRNA ending in the typical stem-loop (SL) structure and substantial amounts of an extended poly(A) mRNA. We show, by the selective removal of either mRNA isoform, that the S-phase-specific H2A.X SL mRNA can generate sufficient histone for deposition onto DNA damage responsive (DDR) chromatin in some cell types. In contrast, when cells have evolved to make predominantly H2A.X poly(A) mRNA they then require de novo H2A.X for efficient DDR. This highlights the importance of differential H2AFX mRNA 3’ end processing for the maintenance of effective DDR To characterise histone mRNA transcription profiles across the cell cycle, HeLa cells were synchronised in S or G1 phases using either aphidicolin or nocodazole, respectively. arrested cells were then subjected to nuclear and cytoplasmic fractionation, followed by protein and RNA isolation. Nuclear and cytoplasmic mRNA was then subjected to poly(A)+ selection, followed by ribosomal RNA depletion of the poly(A)- mRNA. Both poly(A)+ and poly(A)- mRNA were used for RNA-seq library preparation and then subjected to Illumina sequencing.